Molecular Mechanisms of Thyroid Hormone-stimulated Steroidogenesis in Mouse Leydig Tumor Cells

Using a mouse Leydig tumor cell line, we explored the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis. Triiodothyronine (T3) induced a ∼3.6-fold increase in the steady-state level of StAR mRNA which paralleled with those of the acute steroid response (∼4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production was effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displayed the highest affinity of [125I]iodo-T3 binding and was most potent in stimulating StAR mRNA expression. In accordance, T3significantly increased testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression were similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) showed marginal elevation of StAR expression, but coordinately increased T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1 markedly diminished the SF-1 mRNA expression, and concomitantly abolished T3-mediated responses. Noteworthy, T3 augmented the SF-1 mRNA expression while inhibition of the latter by DAX-1 strongly impaired T3 action. Northern hybridization analysis revealed four StAR transcripts which increased 3–6-fold following T3 stimulation. These observations clearly identified a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis.

The acute trophic hormone-responsive steroidogenesis is dependent on mobilization of cholesterol from cellular stores to the mitochondrial inner membrane. This is a rate-limiting and regulated step in steroidogenesis and is dependent on de novo protein synthesis in steroidogenic cells (1,2). Conversion of cholesterol to pregnenolone is the first enzymatic step in steroidogenesis which is catalyzed by the cholesterol side chain cleavage cytochrome P450 enzyme, located in the inner mitochondrial membrane (3)(4)(5). Several factors have been proposed as essential mediators for cholesterol delivery into this site critical for the initiation of steroidogenesis (6). It has been demonstrated that luteinizing hormone, its "superagonist" hCG, 1 and the analog of their second messenger (cAMP), dibutyryl-cAMP (Bt 2 cAMP), cause in MA-10 mouse Leydig tumor cells increased synthesis of a series of 37-, 32-, and 30-kDa proteins, which are closely associated with mitochondria (7)(8)(9). Inhibition of protein synthesis in the hormone-stimulated steroidogenic cells has been shown to decrease their response in steroid biosynthesis. It is known that the inhibitor-sensitive step is present in the mitochondria and that cycloheximide (CHX) has no effect on the activity of cholesterol side chain cleavage complexes or on cholesterol accumulation to the outer mitochondrial membrane (10,11).
A role for thyroid hormones has long been implicated in mammalian testicular and ovarian function (12)(13)(14). Hypothyroidism is associated with abnormalities in sexual function, such as azoospermia, oligozoospermia, and loss of libido and impotence in men, whereas in women it causes irregular menstrual bleedings and impaired fertility due to corpus luteum insufficiency (15). Oppenheimer et al. (16) first demonstrated the presence of nuclear-binding sites for T 3 in the rat testis. It has also been demonstrated that the morphological and functional development of testes is highly dependent on thyroid hormones, especially under regulation of the nuclear T 3 receptor (17,18). In goat (Capra hircus) Leydig cells, T 3 induces a proteinaceous factor, sensitive to the protein synthesis inhibitors actinomycin D or CHX, which is present in the soluble supernatant fractions of 100,000 ϫ g, and is responsible for androgen production (19). It was also found that T 3 augments the stimulatory effect of leutinizing hormone on androgen secretion. In cultured human luteinized granulosa cells, T 3 modulated hCG-induced progesterone secretion, cell proliferation, and cAMP production, whereas it had no effect on medium size follicles (20,21). The mechanisms underlying the T 3 -hCG interaction in mouse Leydig tumor cells have so far remained elusive. Thyroid hormone receptors have also been demon-strated in human granulosa, corpus luteum, and rat granulosa cell nuclei (13,22,23). Immunocytochemical studies with antiserum against the cellular erythroblastosis A/T 3 receptor also provide evidence for the presence of T 3 receptor protein in the rat tissues (24). These data imply that thyroid hormones are involved in reproduction, and the effects are known to be absolutely dependent on de novo protein synthesis. However, nothing is still known about the nature of the T 3 -induced protein(s) in the regulation of steroidogenesis.
Recently, an leutinizing hormone-induced 30-kDa mitochondrial factor, named the StAR protein, has been purified and cloned from the MA-10 cells, and it has the necessary properties of inducing steroidogenesis (25). The cDNA clones for StAR have also been isolated from the human, rat, and cow, and they exhibit a high degree of homology (26 -28). The StAR gene is known to encode a 37-kDa nonphosphorylated protein that is processed to a 30-kDa mature form via four intermediates, with half-lives of 3-5 min in the mitochondria (29,30). Northern hybridization analysis revealed the presence of three StAR transcripts in the mouse (3.4, 2.7, and 1.6 kilobases) and human (7.4, 4.4, and 1.6 kilobases), and two in the cow (3.0 and 1.8 kilobases) (25,26,28). In MA-10 mouse Leydig tumor cells, the cAMP-mediated stimulation of steroidogenesis is well correlated with StAR mRNA expression and StAR protein synthesis (29).
Despite the potential involvement of thyroid hormones in steroidogenesis, their precise mode of action remain to be determined. The important role of StAR in steroidogenesis prompted us to examine its involvement in the thyroid hormone stimulation, and consequently in the mechanism of action by employing the orphan nuclear receptors SF-1 and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome). Recent studies implicate that SF-1 and DAX-1 play a key role in adrenal and gonadal differentiation, development, and function (31)(32)(33). Impaired adrenal development and function, associated with hypogonadotropic hypogonadism, appear due to mutations of these two nuclear receptors. The complex endocrine phenotypes with the SF-1 and DAX-1 mutations point strongly to their interactions in a hierarchial pathway, and they may activate the target genes in a cooperative fashion (34). Conspicuously, SF-1 knock-out mice completely lack adrenal glands and gonads despite normal embryonic levels of serum corticosterone (35). It is apparent that understanding the involvement of thyroid hormones into the regulation of steroidogenesis requires exploration of the above two transcription factors.
In light of these observations, we found it important to examine the physiological relationship of thyroid hormone action and StAR gene expression during steroidogenesis, using mouse Leydig tumor cells (mLTC-1, Ref. 36) as the experimental model. The current findings demonstrate for the first time that thyroid hormones, through induction of the SF-1-mediated StAR gene expression, play a major role in the regulation of steroidogenesis.
Transfections were carried out at 65-75% confluency of the cells by using FuGENE 6 transfection reagent (Boehringer-Mannheim GmbH, Mannheim, Germany) under optimized conditions according to the instructions of the manufacturer. Briefly, FuGENE 6 transfection reagent was diluted in serum-free Waymouth's medium and incubated at room temperature to a final volume of 300 l. Two g of pCMV119 ϩ -SF-1 construct, obtained from Dr. K. L. Parker (Duke University Medical Center, Durham, NC), and 2 g of pBKCMV-hDAX-1 construct, obtained from Dr. R. Yu (Northwestern University Medical School, Chicago, IL), or their combination (1:1), were used for transfections. Two g of a ␤-galactosidase expression vector, pSV-␤-galactosidase (Promega, Madison, WI) were used as an internal control of tranfection efficiency. FuGENE 6-DNA complex following 15 min incubation at room temperature were distributed dropwise to the plate containing 3 ml of regular medium.
RNA Extraction, and Quantitative Reverse Transcription and Polymerase Chain Reaction-Total RNA was isolated from control and stimulated cells by the acid guanidinium thiocyanate/phenol/chloroform extraction method of Chomczynski and Sacchi (40). The purity of the extracted RNA was determined by scanning with spectrophotometer at wavelength 220 -320 nm.
The isolation and amplification of the mouse (mLTC-1) StAR cDNA were carried out by engineering primers from the mouse StAR cDNA sequence (25). The sense primer, 5Ј-GACCTTGAAAGGCTCAGGAA-GAAC-3Ј, and the antisense primer, 5Ј-TAGCTGAAGATGGACAGACT-TGC-3Ј, spanned bases Ϫ51 to Ϫ27 and 931 to 908, respectively, in relation to the first base of the translation initiation codon. To evaluate the potential variation in RT-PCR efficiency, an internal control, a 395-bp fragment of the L19 ribosomal protein gene was coamplified in each sample, using as sense primer 5Ј-GAAATCGCCAATGCCAACT-C-3Ј and as antisense primer 5Ј-TCTTAGACCTGCGAGCCTCA-3Ј.
RT and PCR of the target genes were run sequentially in the same assay tube, as described previously (41). Briefly, equal amounts of total RNA from the different experimental groups (2 g/sample) were reverse transcribed using avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland) and the antisense primers. The cDNAs generated were further amplified by PCR using the primer pairs mentioned above. The total reaction volume was 50 l which contained 1 nmol/liter of each oligo primer, 200 mol/liter of a deoxy-NTP mixtures including [␣-32 P]CTP, 20 units of RNasin, 12.5 units of avian myeloblastosis virus-reverse transcriptase-RT, and 2.5 units of Dynazyme-DNA polymerase in 1 ϫ PCR buffer (10 mmol/liter Tris-HCl, 50 mmol/ liter KCl, 1.5 mmol/liter MgCl 2 , and 0.1% Triton X-100, pH 8.8) (Finnzymes). The reaction was initiated at 50°C for 15 min (RT) followed by denaturation at 97°C for 5 min. Then the PCR was run with a variable number of cycles of amplification defined by denaturation at 96°C for 1.5 min, annealing at 55°C for 1.5 min, and extension at 72°C for 3 min (PTC-200, Peltier Thermal Cycler, MJ Research, Watertown, MA). The number of PCR cycles examined was 16 to 40, and 16 cycles were chosen for further analysis (data not shown). A final cycle of extension at 72°C for 15 min was included. To examine the PCR products, a 25-l aliquot of each reaction was analyzed by gel electrophoresis on a 1.2% agarose gel. The molecular sizes of the amplified products (StAR and L19) were determined by comparison with the molecular weight markers run in parallel with RT-PCR products. The gels were then vacuum dried and exposed to Kodak x-ray films (XAR-5, Eastman Kodak, Rochester, NY) at 4°C for 1 to 3 h, and autoradiograms were analyzed for StAR mRNA expression. The relative levels of different signals were quantitated by densitometry (Tina 2.0 Package, Straubenhardt, Germany).
Generation of a StAR Competitor (StAR-2) and Competitive RT-PCR-For the generation of a competitor, full-length StAR cDNA pro-duced by RT-PCR, as described previously, was subcloned into the pGEM T-vector following instructions of the manufacturer (Promega). The identity of the inserted fragment was confirmed by sequencing with fluorescent dye termination reaction (Prism Ready Reaction Dye Termination Cycle Sequencing Kit) using an automated sequencer (Perkin-Elmer).
Taking advantage of the presence of two AvaI sites in the StAR cDNA, the subcloned product was subjected to restriction endonuclease digestion, followed by separation in 1.2% agarose gel electrophoresis. The existing AvaI fragment of 407-bp from the full-length StAR cDNA was excised from the gel. The remaining fragments (StAR cDNA subcloned into pGEM T-vector) were subjected to blunt-end ligation by the Klenow fragment of DNA polymerase I and the strand was recircularized by T4 DNA ligase, therefore generating a StAR cDNA containing a 407-bp deletion, inserted into the cloning site of the pGEM T-vector. Transcription of the ApaI linearized template by means of the Sp6 RNA polymerase (Promega) generates a sense cRNA of approximately 570 bp length, which was used as the StAR competitor (StAR-2, Fig. 1).
To confirm the validity and reliability of the quantitative results obtained by RT-PCR, StAR mRNA levels were further assessed by competitive PCR. For this procedure, decreasing concentrations (800 -1.56 ng) of the StAR competitor (StAR-2) cRNA were amplified together with a fixed amount of target RNAs (2 g of total RNA from control and T 3 stimulated samples), using the same primer pairs as above. Aliquots of 25 l of the RT-PCR products were analyzed and the levels of StAR mRNA were evaluated as described above.
Northern Hybridization Analysis-Twenty micrograms of the total RNA isolated from control and treated groups were resolved on 1.2% denaturing formaldehyde agarose gel and transferred onto Hybond-N ϩ nylon membrane (Amersham Int., Aylesbury, UK) by employing the capillary transfer method. The StAR and SF-1 cDNA probes were labeled with [␣-32 P]dCTP (1800 Ci/mmol) using the Prime-a-Gene labeling method (Promega). The labeled probes were purified by using Sephadex G-50 nick columns (Pharmacia). Prehybridization and hybridization were carried out under stringent conditions as described previously (42). After hybridization, the membranes were washed twice at room temperature for 20 min with 2 ϫ SSC containing 0.1% SDS, followed by 2 h at 42°C with 0.1 ϫ SSC and 0.1% SDS until removal of the background counts. To examine the variation in StAR and SF-1 mRNA levels, the membranes were subjected to rehybridization with a cDNA probe of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membranes were then exposed to x-ray films (Kodak XAR-5) for 60 -72 h at Ϫ80°C. The relative mRNA levels of StAR in different transcripts, SF-1, and GAPDH expression were quantitated as above.
[ 125 I]Iodo-T 3 (1080 -1320 Ci/g) binding to the mLTC-1 cell nuclear membrane preparations was performed according to the procedure described earlier (19,44), slightly modified for optimum binding. Briefly, a nuclear membrane preparation (25 g of DNA/incubation) was incubated with a fixed concentration of [ 125 I]iodo-T 3 (100,000 cpm) either in the absence (total) or presence of 600-fold excess of unlabeled T 3 (nonspecific) in a final volume of 400 l. For the competitive inhibition studies, 25 g of DNA were incubated with 100,000 cpm of [ 125 I]iodo-T 3 together with varying concentrations of the unlabeled hormone (1.5-1,500 pmol/liter). The incubation was carried out at 37°C for 2 h, and terminated by adding 2 ml of ice-cold 40% polyethylene glycol (M r 6,000). After centrifugation at 3,000 rpm for 20 min at 4°C, the supernatant was discarded and the pellet washed twice with the incubation buffer, and finally radioactivity in the pellet was determined in a ␥-counter (1260 Multigamma II, LKB Wallac, Turku, Finland).
Determination of Progesterone (P) and Testosterone (T)-The concentrations of P and T in the media were assessed, after extraction with diethyl ether, with specific RIAs as described previously (45,46).
Statistical Analysis-The data are presented as the mean Ϯ S.E. from representative cultures carried out in triplicate or quadruplicate. Statistical analysis was performed using the Statview program following ANOVA (FisherЈs protected least significant differences test) as noted. A p value less than 0.05 was considered statistically significant.

T 3 -induced P Production Requires Protein Synthesis-
The induction of P production by T 3 was evaluated for the dependence of on-going protein synthesis. The inhibitors of protein synthesis, actinomycin D and CHX (10 mg/liter each), the former preventing incorporation of methionine into protein and the latter inhibiting RNA synthesis, were examined separately. The cells stimulated with T 3 (37.5 pmol/liter) for 8 h showed a significant (4.0 Ϯ 0.2-fold) increase in P production (Fig. 2). Treatment with actinomycin D or CHX remarkably diminished (p Ͻ 0.0001) T 3 -induced P production, suggesting that the T 3 action is dependent on intact transcription and translation of the target cells. Actinomycin D or CHX alone exhibited no effect on the basal P production. Fig. 2 represent the time-dependent increase in steady-state levels of StAR mRNA and P production in response to T 3 (37.5 pmol/liter) stimulation. The T 3 -induced StAR mRNA expression was significant (p Ͻ 0.05) as early as 30 min, and the magnitude of the response gradually increased up to a 3.6 Ϯ 0.4-fold maximum by 8 -10 h (Fig. 3A). The StAR mRNA levels declined sharply thereafter, reaching another plateau between 16 and 24 h. The cAMP analog 8-Br-cAMP, a well known inducer of the StAR gene expression through activation of the PKA signal transduction pathway (29,47,48), was used as a positive control stimulus. It could be clearly seen that a 4-h stimulation with 8-Br-cAMP (1 mmol/liter) elevated the StAR mRNA and P levels about 3.4-fold, which was comparable to that achieved by T 3 stimulation in 4 h. The P levels at each time point were determined and expressed as fold increases, and they paralleled those occurring in StAR mRNA expression (Fig. 3B). The accumulation of P in the medium was significant within 1 h FIG. 1. Schematic representation of the mouse StAR and StAR-2 cDNAs. StAR-2 cDNA was generated from the full-length mouse StAR cDNA by deleting a 407-bp fragment (hatched rectangle) using two AvaI sites. This shortened cDNA fragment was used as a template to generate the cRNA competitor (StAR-2) by means of Sp6 RNA polymerase. The corresponding positions of the sense and antisense PCR primers in the cRNA are illustrated by arrows.

Effect of T 3 on StAR mRNA Expression and P Production by mLTC-1 Cells-The results summarized in
(p Ͻ 0.0001), and it gradually increased reaching a plateau between 7 and 9 h, and then followed the pattern of StAR expression. The concordant time courses of the two T 3 responses demonstrate a direct correlation between StAR mRNA expression and P production.
Since the StAR mRNA expression and P production are clearly stimulated by T 3 in a time-dependent manner, we next examined the dose-response pattern of the T 3 effects. The results presented in Fig. 4 show that when the cells were stimulated with increasing concentrations of T 3 (0.015-1500 pmol/ liter) for 8 h, a dose-dependent effect was observed on StAR mRNA expression and P production. T 3 induced an increasing response in StAR mRNA levels, as determined by quantitative RT-PCR (Fig. 4A). Increased StAR mRNA expression was detected (p Ͻ 0.05) at concentrations Ն 1.5 pmol/liter, the halfmaximal stimulation occurring at 16.5 pmol/liter, and maximum increase at doses Ն 37.5 pmol/liter of T 3 (Fig. 4B). The dose-response pattern of P production was similar with significant response at T 3 level Ն 1.5 pmol/liter (Fig. 4C), further indicating a direct link between the T 3 effect on steroidogenesis and StAR expression.
Competitive RT-PCR Approach for Quantitation of the T 3induced StAR mRNA Expression-To verify the accuracy and validity of the quantitative RT-PCR results, we also examined the competitive PCR approach. A homologous cRNA (StAR-2) was included into the RT-PCR reaction to determine the sensitivity of our assay system in competitive conditions. The results of the competitive PCR are presented in Fig. 5, where fixed amounts of total RNA (2 g) either from control or T 3 (37.5 pmol/liter)-stimulated samples were allowed to compete with varying quantities (800 -1.56 ng) of the cRNA competitor (StAR-2). As expected, amplification of decreasing amounts of the competitor with fixed amounts of total RNA provided signals of decreasing intensity. Conversely, the StAR signals gradually decreased as the concentrations of competitor increased, indicating a specific nature of the competitive PCR reactions (Fig. 5A). The ratios of intensities of the StAR/StAR-2 amplification products in both cases were corrected for the constant intensity of L19 band as illustrated on the graph of Fig. 5B. It could be clearly seen that T 3 induced about 3.6-fold increase in StAR mRNA expression as compared with control. The experiment further confirmed the previous results obtained by quantitative RT-PCR, indicating that T 3 exerts a specific action on StAR mRNA expression.  FIG . 3. The time response pattern of T 3 -stimulated StAR mRNA expression and P production of the mLTC-1 cells. Total RNA was extracted from control and T 3 (37.5 pmol/liter)-treated cells and subjected to RT-PCR analysis as described under "Experimental Procedures." A 395-bp fragment of the ribosomal protein L19 gene was coamplified in each sample to correct for the variation in RT-PCR efficiency. The RT-PCR products were resolved in 1.2% agarose gels, which were dried, and exposed to x-ray films. A representative autoradiogram showing the time response (0 -24 h) of the T 3 -induced StAR mRNA expression, and a 4-h response to 8-Br-cAMP treatment (1 mmol/liter) was used as the positive control (Panel A). Panel B shows the arbitrary densitometric units of the StAR mRNA responses, and the P production of the same samples, both expressed as fold-increases over the control (CON) level. The arbitrary densitometric unit value of each band was corrected with the corresponding L19 bands. The results are the mean Ϯ S.E. of three independent experiments in duplicate. The asterisks represent significance of differences in comparison to control; *, p Ͻ 0.05; ****, p Ͻ 0.0001; 3, the first time point with significant elevation from control; 4, significant reduction in comparison to maximal T 3 stimulation.
To corroborate the observed effect of hCG and T 3 in StAR expression we also determined their response in P production. Stimulation of cells with these hormones at ED 50 doses, hCG (0.3 pmol/liter), and T 3 (16.5 pmol/liter), either separately or in combination, clearly increased P accumulation and followed a similar pattern as evidenced with StAR mRNA expression (Fig.  6C). These data further suggest a close coordination between the stimulated levels of StAR expression and P production.
Specificity of Thyroid Hormone Binding to Nuclear Membrane Preparations and on StAR mRNA Expression-To examine the presence of thyroid hormone receptors, competitive inhibition experiments were carried out under optimized conditions with nuclear membrane preparations of the mLTC-1 cells. Fig. 7A shows that at increasing concentrations (1.5-1500 pmol/liter) of unlabeled T 3 , triiodothyroacetic acid (TRIAC), and thyroxine (T 4 ), T 3 demonstrated the highest affinity for these binding sites.
The results presented so far clearly demonstrate that T 3 modulates StAR gene expression and P production. We next examined whether other thyroid hormones, i.e. TRIAC and T 4 , would induce StAR mRNA expression. As shown in Fig. 7, B and C, all three thyroid hormones exerted significant increases on StAR mRNA levels in 8 h; however, TRIAC and T 4 were less potent than T 3 .

Effect of T 3 on Dispersed Leydig
Cell T Production-To assess functional relevance of the thyroid hormone effects observed with the murine Leydig tumor cells, adult mouse primary Leydig cells were stimulated in the absence or presence of two concentrations of T 3 (7.5 and 75 pmol/liter) and hCG (0.7 pmol/liter) for 8 h. As shown in Fig. 8, T 3 at both doses significantly augmented the T production, which was 1.19 Ϯ 0.07and 1.8 Ϯ 0.21-fold higher, respectively, over nonstimulated cells. The cells were simultaneously incubated with hCG (0.7 pmol/liter) for comparison, which also showed a clear stimulation of T production.
Northern Hybridization Analysis of StAR mRNA and T 3 Stimulation-Besides RT-PCR, it was also possible to show the effect of T 3 on StAR expression by Northern blot analysis. Total RNA was extracted from control and T 3 (37.5 pmol/liter)-stimulated mLTC-1 cells, and probed with the StAR cDNA. The full-length mouse StAR cDNA probe hybridized with two major RNA transcripts of 3.4 and 1.6 kb in size, of which the latter corresponds to the functional StAR protein (Fig. 9). In addition, two minor transcripts of 2.7 and 1.4 kb sizes were detected when the membranes were exposed for longer time (72 h). The intensities of all the transcripts increased in parallel, 3-6-fold, with T 3 stimulation. cells. In contrast, cells expressing DAX-1 displayed remarkably diminished (p Ͻ 0.0001) T 3 -induced StAR expression and P production. The magnitude of the SF-1-mediated response was attenuated following T 3 stimulation when the cells were cotransfected with SF-1 and DAX-1 (Fig. 10), indicating that SF-1 is an essential regulator of thyroid hormone-mediated StAR gene expression. The accumulation of P levels in the media from non-transfected, SF-1, DAX-1, and SF-1 ϩ DAX-1 expressing cells with increasing concentrations of T 3 (0.15-150 pmol/liter) exhibited a dose-dependent response, while DAX-1 drastically inhibited the augmenting effect of T 3 on P production (Fig. 11).

Expression of SF-1 and DAX-1 in mLTC-1 Cells, and Their Correlation to the Levels of T 3 -induced StAR Expression and P
Effect of SF-1 and DAX-1 Expression on the Levels of Endogenous and T 3 -stimulated SF-1 mRNA-Northern hybridization analysis revealed that the level of SF-1 mRNA in mLTC-1 cells increased significantly (p Ͻ 0.0001) upon transfection of the SF-1 expression plasmid. In contrast, the SF-1 levels were significantly suppressed by DAX-1 and SF-1 ϩ DAX-1 co-expression (Fig. 12). Given the direct link between StAR expression and steroidogenesis, we examined the T 3 -stimulated SF-1 mRNA expression, which clearly showed a dose-dependent elevation, with a 2.9 Ϯ 0.3-fold maximum in comparison to nonstimulated cells, favoring the indirect steroidogenic effect of T 3 rather than a direct one (Fig. 13). DISCUSSION The acute production of steroid hormones is initiated by translocation of cholesterol from the outer to the inner mitochondrial membrane, an event that has recently been found to be mediated by the StAR protein. StAR is a novel 30-kDa protein associated with mitochondria, and its level is rapidly induced in response to trophic hormone or cAMP stimulation in steroidogenic cells (8). The present experiments were designed The nuclei were prepared from the cells and subjected to the nuclear receptor binding assay as described under "Experimental Procedures." The results are the mean Ϯ S.E. of four experiments in triplicate. Panels B and C, induction of StAR mRNA expression by thyroid hormones. Total RNA was extracted from different groups of mLTC-1 cells after 8 h incubation without (CON) or with T 3 , TRIAC, and T 4 (each 37.5 pmol/liter). The RT-PCR analyses were carried out using 2 g of total RNA from each group, and the RT-PCR products were analyzed as described under "Experimental Procedures" and in the legend of Fig. 3. A representative autoradiogram demonstrating the action of thyroid hormones on StAR mRNA levels is presented in Panel B. The intensities of the StAR mRNA amplicons (arbitrary densitometric units) after correction for the intensity of the corresponding L19 bands, are presented in Panel C. The values are the mean Ϯ S.E. (n ϭ 4). Different letters above the bars indicate that these groups are significantly different at a level p Ͻ0.001.
to explore the regulation of the StAR gene expression by thyroid hormones, and in particular by T 3 , biologically the most potent thyroid hormone. Our data demonstrate the presence of thyroid hormone receptors in nuclei of the mLTC-1 mouse Leydig cells where T 3 increases the steady-state levels of StAR mRNA and steroid production. The most intriguing results presented here are the parallel temporal and dosage patterns of the T 3 -induced StAR expression and steroid production which provide evidence that T 3 stimulation of the StAR gene expression and steroidogenesis are linked to a common regulatory cascade. In addition, the T 3 and hCG effects on StAR mRNA expression and P production appear to be additive in these cells. The physiological relevance of these findings was assessed by T 3 -stimulated T production of primary mouse Leydig cells. Collectively, to our knowledge, this is the first demonstration of involvement of thyroid hormones in StAR gene expression, which may provide the mechanism for the known importance of thyroid function for that of the gonads.
The classical mechanism of stimulation of steroidogenesis occurs through trophic hormone-stimulated cAMP production, and concerns both acute and chronic effects on steroid production (25,29). This regulatory cascade involves the transport of cholesterol to mitochondria, which is the first step in the biosynthesis of all steroid hormones. It has been demonstrated that during cholesterol transport both outer and inner mitochondrial membranes become closely associated and form "contact sites" which are dependent on the transfer of phospholipids (49 -51). The increase in contact sites is closely associated with the stimulation of steroidogenesis. The trophic hormone or cAMP-induced StAR gene transcription requires on-going protein synthesis which is responsible for the acute regulation of steroidogenesis (1,32). Clark et al. (29) have demonstrated that in MA-10 cells, Bt 2 cAMP coordinately stimulates StAR protein and StAR mRNA expression which were consistent with stimulated steroidogenesis. Our results document that T 3 promotes StAR gene expression and P production in a time-and dosedependent manner.
The hormone-induced StAR protein is processed from a short-lived 37-kDa precursor protein that is sensitive to cycloheximide (25,52). Epstein and Orme-Johnson (30) isolated a mitochondrial phosphoprotein in adrenal glomerulosa cells that fulfills the criteria of a regulator of steroidogenesis and resembles the StAR protein. Recent studies also implicate that phosphorylation of the serine residue at codon 194/195 of StAR modulates steroidogenic activity of the StAR protein (53). It has been shown that blockade of the StAR protein synthesis at the translational level prevents the cholesterol transport into adrenal glomerulosa cells (54). In mechanically dispersed testicular Leydig cells, T 3 induced significant androgen produc-tion by generating a 52-kDa soluble protein, also sensitive to the protein synthesis inhibitors (19). Our findings clearly demonstrate that T 3 stimulation of P production in mLTC-1 cells is inhibited by actinomycin D or CHX, indicating the involvement of acute protein synthesis in the regulation of steroidogenesis, which seems to be mediated by StAR protein. Moreover, T 3 markedly and in additive fashion augmented the hCG-induced expression of StAR mRNA and P production. This is in keeping with the different mechanisms of action of T 3 and leutinizing hormone/hCG, and suggest that these effectors employ different cis-and/or trans-activators of the StAR gene.
A pertinent question arising from the T 3 -induced StAR expression concerns its biological relevance. To explore this particular aspect, other thyroid hormones, TRIAC and T 4 , were examined in the same manner for StAR mRNA expression and competitive nuclear binding studies. The results also demonstrate the specificity for T 3 of the thyroid hormone-binding sites in mLTC-1 cell nuclei, whereas TRIAC and T 4 were shown to induce weaker responses in both StAR mRNA expression and in inhibition of [ 125 I]iodo-T 3 binding. However, a single class of high-affinity receptors was observed in these cells. The affinity (K d Х 0.486 nmol/liter) of T 3 -binding sites (not illustrated) to the nuclear preparations of mLTC-1 cells was comparable to that reported in conventional target tissues for thyroid hormones (15,19,55). The physiological response of thyroid hormone was assessed in purified adult mouse testicular Leydig cells, where T 3 and hCG both were equipotent in inducing T production.
The crucial role of StAR protein in the regulation of steroi- dogenesis has been demonstrated in StAR knock-out mice (56), and in patients suffering from lipoid congenital adrenal hyperplasia, which was found to be due to mutations in the StAR gene (57). Furthermore, Northern and Western analyses have demonstrated that expression of the StAR gene is involved in regulating steroidogenesis in MA-10 cells (27,29). Our present model of the mechanisms of T 3 -induced StAR mRNA expression and steroidogenesis reflects similar phenomena, indicating that T 3 -induced StAR protein is directly involved in the acute regulation of P production. In addition, four StAR transcripts were detected in the mLTC-1 cells, two major (3.4 and 1.6 kb) and two minor (2.7 and 1.4 kb) ones, and all of them were increased upon T 3 stimulation. The molecular sizes of the different transcripts corresponded to those previously reported in different species (25)(26)(27)(28). It is also possible that the minor and inconsistent band at 1.4 kb is a splice variant of the StAR gene with differential polyadenylation signal or length of the poly(A) tail.
With respect to the interaction of T 3 with StAR expression, its mechanism of action is of considerable interest. Stocco and collaborators (29,52) and others (27,54) have provided ample evidence that StAR expression is predominantly regulated by the cAMP second messenger system, although other factor(s) might also be involved. There is a conspicuous lack of cAMP response elements in the mouse and human StAR promoter sequences (47,58). This is not surprising since it seems to be a common feature of many cAMP-regulated genes, including the cytochrome P450 steroid hydroxylase genes (59). It has been implicated that the orphan nuclear receptors SF-1 and DAX-1 have pivotal roles in the regulation of reproductive endocrine functions at multiple levels during development and differentiation, including the expression of steroidogenic enzymes (33,60,61). Studies also indicate that the mutations associated with DAX-1 and SF-1 cause adrenocortical insufficiency and gonadal abnormalities associated with hypogonadotropic hypogonadism (31). In addition, DAX-1 binds to DNA hairpin structures and suppresses the transcriptional activity of SF-1 and StAR in Y-1 adrenocortical tumor cells (62,63). On the other hand, the SF-1 knock-out mice provided intriguing information pertaining to the role of SF-1 as a key regulator of endocrine function and expression of the steroidogenic enzymes (35). The nucleotide sequences of the 5Ј-flanking region of the mouse StAR gene (3.6 kb) revealed the presence of two SF-1binding sites at positions Ϫ135 and Ϫ42, and one DAX-1binding site at Ϫ24 position. The importance of the SF-1binding sites for StAR expression has been further confirmed by the deletion of those sequences from the mouse StAR promoter, which significantly diminished its activity (47). Two potential binding sites are also located at positions Ϫ890 and Ϫ42 for the nuclear hormone receptors which resembles the SF-1 consensus motif, whereas neither the human nor the mouse StAR promoter sequences contain specific thyroid hormone receptor recognition sites (47,58). For this reasons, and since T 3 augmented the StAR expression of the cells constitutively over-expressing SF-1, T 3 -stimulated SF-1 expression does not totally explain its effect on StAR, and additional facts are apparently involved.
The cells expressing SF-1 did not only respond with increased StAR mRNA expression and P production, but these levels of expression were also under the regulation of T 3 . In contrast, DAX-1 expression drastically repressed the potential effects of T 3 on StAR gene expression and P production, whereas it markedly diminished the SF-1-mediated expression. Strikingly, in a dose-dependent manner, T 3 stimulation significantly elevated the levels of SF-1 mRNA expression, whereas DAX-1 profoundly inhibited the endogenous SF-1 level. These data strongly suggest that T 3 has no direct action on StAR gene in the regulation of steroidogenesis, rather an indirect effect through the modulation of SF-1 expression. The precise mechanisms of thyroid hormone action in steroidogenesis may be multifactorial, and remains to be established. On the basis of the present observations, it is tempting to propose that the mechanism of thyroid hormone action on StAR gene expression, which consequently regulates steroidogenesis, is at least in part mediated by SF-1. The regulation of SF-1 function together with the post-translational modifications, i.e. phosphorylation and the interactions of SF-1 with coactivator(s) involved in T 3 -induced StAR gene expression, will be an area of obvious interest in our future investigations.